1. Field of the Invention
The present invention relates to the use of fibroblast growth factors as therapeutic agents for the prevention and treatment of pathological conditions involving bone tissue, for example, osteoporosis, Paget's disease, osteopetrosis, and periodontal disease and fracture repair, and healing of bone defects.
2. Background of the Invention
Living bone tissue is continuously being replenished by the processes of resorption and deposition of bone matrix and minerals. This temporally and spatially coupled process, termed bone remodeling, is accomplished largely by two cell populations, the osteoclasts and osteoblasts. The remodeling process is initiated when osteoclasts are recruited from the bone marrow or the circulation to the bone surface and remove a disk-shaped packet of bone. The bone matrix and mineral is subsequently replaced by a team of osteoblasts recruited to the resorbed bone surface from the bone marrow. Among the pathological conditions associated with abnormal bone cell function is osteoporosis, a diseased characterized by reduced amounts of bone (osteopenia) and increased bone fragility. These changes can be the result of increased recruitment and activity of osteoclasts, often in combination with reduced recruitment or activity of osteoblasts. It is believed that the development of excess or deficient populations of osteoclasts or osteoblasts may result from a corresponding lack or excess of specific protein factors called cytokines.
Cytokines have been identified by their biological characteristics and their unique amino acid sequences. Each cytokine presents a unique spectrum of characteristics that distinguish it from other cytokines. In general, the cytokines stimulate the growth and/or differentiation of specific types of cells. Some cytoines can also target cancerous cells for destruction. Examples of cytokines include granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF), interleukin-1 beta, interleukin-3, interleukin-6, interferon-gamma, tumor necrosis factor, lymphotoxin, leukemia inhibitory factor, fibroblast growth factors, transforming growth factor-alpha and transforming growth factor-beta.
Many of the known cytokines stimulate or inhibit blood cells. Several growth regulatory cytokines, such as M-CSF, transforming growth factor alpha, interleukin-1 and tumor necrosis factor, have been shown to stimulate marrow mononuclear cell proliferation. Although cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF) and interleukin-6 (IL-6) may influence osteoclast formation and differentiation (Mundy (1990) Trends Endo. Metab. 1:307-311), these factors are not specific osteoclast growth regulatory factors.
Although there is much information available on the factors that influence the breakdown and resorption of bone, information is more limited on factors that can actually stimulate the formation of new bone. Bone itself contains factors that have the capacity for stimulating the growth and/or differentiation of bone cells. Thus, extracts of bone tissue contain not only structural proteins that are responsible for maintaining the structural integrity of bone, but also biologically active bone growth factors that stimulate bone cells to proliferate. Among the growth factors found in bone that are known to stimulate proliferation of bone cells are transforming growth factor .beta., the insulin-like growth factors (insulin-like growth factor I and insulin-like growth factor II), basic fibroblast growth factor (bFGF) and bone morphogenetic proteins (BMPs). These factors also cause proliferation of non-bone cell types.
The fibroblast growth factor (FGF) family is comprised of at least 9 structurally related proteins (FGFs 1-9), whose best known members are acidic FGF (aFGF; FGF-1) and basic FGF (bFGF; FGF-2). Members of this family stimulate mitogenesis in most cells derived from the mesoderm and neuroectoderm and influence other biological processes, including angiogenesis, neurite extension, neuronal survival, and myoblast differentiation. In general, FGFs have a high affinity for heparin (prior to resolution of their nomenclature, some FGFs were referred to as heparin-binding growth factors -1, -2, etc.), and many, but not all, are mitogens for fibroblasts. The members of the FGF family possess roughly 25-55% amino acid sequence identity within a core sequence and some FGFs possess significant extensions, either C-terminal, N-terminal, or both, outside of this core sequence. This structural homology suggests that the 9 different genes encoding known FGFs may be derived from a common, ancestral gene.
In addition to the 9 known members of the FGF family, additional complexity results from the generation of several molecular forms of FGF from a single gene. For example, the primary translation product of aFGF (FGF-1) consists of 155 residues. However, the longest form of FGF-1 found in a natural source (e.g., bovine brain) consists of 154 residues. This 154 residue form of FGF-1 lacks the NH2-terminal methionine of the 155 residue form and has an acetylated amino terminus. Proteolytic processing in vivo or during purification generates smaller active forms of FGF-1 in which either the amino-terminal 15 (des 1-15) or 21 (des 1-21) amino acids are deleted. As defined herein, FGF-1 refers to the 154 residue form of FGF-1 and shorter, biologically active forms thereof, such as the above described forms deleted of the amino-terminal 15 (des 1-15) or 21 (des 1-21) amino acids. Historically, the 154 residue form of FGF-1 was termed .beta.-endothelial cell growth factor (.beta.-ECGF), the des 1-15 form was termed aFGF, and the des 1-21 form was termed .alpha.-ECGF. Prior to standardization of the terminology for this group of growth factors, several additional terms were also applied to the same protein, including eye derived growth factor and heparin binding growth factor 1. Similar forms of bFGF (FGF-2) have also been described. In addition to cleaved forms, extended forms of bFGF have also been described, resulting from initiation of translation at several different GTG codons located upstream of the ATG translation initiation codon which generates the 155 residue form of bFGF. All of these alternative forms of the FGFs contain the core region of structural homology which defines the FGF family.
Reported Developments
An osteogenic role for bFGF was suggested based on in vitro studies (Hauschka et al., J. Biol. Chem 261:12665-12674, 1986; Globus et al., Endocrinology 123:98-105, 1988; Canalis et al., J. Clin. Invest. 81:1572-1577, 1988; McCarthy et al., Endocrinology 125:2118-2126, 1989; Noff et al., FEBS Lett., 250:619-621, 1989). However, there has been only one report of in vivo administration of bFGF (Mayahara et al., Growth Factors 9:73-80, 1993). Intravenous administration of human bFGF to rats demonstrated only endosteal new bone formation. No increase in periosteal bone formation was evidenced. Similar systemic osteogenic potential was seen after intravenous administration of human aFGF.